Fig 2: HPSE promotes the proliferation of CRC cells in vitro. (A) Crispr-Cas9 technology was used to knockdown HPSE expression in SW480 cells and the expression of HPSE was determined by western blotting. (B,C) SW620 cells were infected HPSE lentiviral activation particles and HPSE expression was determined by (B) qRT-PCR and (C) western blotting. The proliferation of (D) SW480 cells and (E) SW620 cells was determined by the CCK-8 assay (n = 3). The OD450 value was assessed at 1, 2, 3, 4, 5, and 6 days respectively. *p < 0.001.

Fig 3: Knockdown of HPSE inhibits CRC cells invasion in vitro and liver metastasis in vivo. (A) An invasion assay was performed in a Matrigel-coated transwell chamber; representative images of invasion assay are shown. (B) The invasive ability of SW480 cells was inhibited by HPSE knockdown (n = 5). (C) SW480 cells were injected into the spleens of BALB/c mice. After 6 weeks, the mice were sacrificed, and their livers were fixed. Microscopic liver metastases were detected by H&E staining. (D) The metastatic foci were counted (n = 4). HPSE knockdown in SW480 cells resulted in fewer metastatic foci in the liver. *p < 0.05, ***p < 0.001.

Fig 4: HPSE promotes the proliferation of CRC cells in vivo. Cells were injected subcutaneously into the back of BALB/c nude mice. Mice were sacrificed at 3 weeks after transplantation. The xenografts were excised and weighted. (A) Knockdown of HPSE expression inhibited tumorigenicity of SW480 cells in BALB/c nude mice (n = 6). The weight of tumors originating from SW480-NC is larger than that arising from SW480-KD1 and SW480-KD2 cells (left panel). A representative photograph of tumor size is shown (right panel). (B) Overexpression of HPSE in SW620 cells promoted the growth of mouse xenograft tumors (n = 6). Scale bar, 1 cm. Immunohistochemistry analysis of HPSE and Ki-67 expression in xenografts originating from (C) SW480 and (D) SW620 cells. Scale bar, 100 µm. Quantification of tumor cell proliferation in (E) SW480 and (F) SW620 xenografts using Ki-67 staining (n = 6). Data are expressed as Ki-67 positive tumor cells as percentage of total tumor cells. *p < 0.05, ***p < 0.001.

Fig 5: HPSE expression in CRC cells. (A) Quantification of HPSE mRNA expression in different CRC cells. The expression of HPSE was normalized to ß-actin. Results are shown as the fold change of CRC cells relative to SW620 cells. Results are representative of three independent experiments and are presented as the mean ± SEM. (B) Western blot analysis of HPSE expression in whole-cell lysates of CRC cells. SW480 and HCT116 cells exhibited higher expression of HPSE compared to the other cell lines. (C) Immunofluorescence staining demonstrated that HPSE was mainly localized in the cytoplasm of CRC cells. Green indicates HPSE and blue indicates DAPI. Scale bar, 20 µm.